High-throughput assay for small molecules targeting the Type I efflux pump of Xylella fastidiosa.
D. GABRIEL (1), M. Jain (1), L. Fleites (1), S. Zhang (1) (1) University of Florida, U.S.A.

 Type I secretion (T1S) by Xylella fastidiosa (Xf), causal agent of Pierce's Disease (PD), is required for pathogenicity, and T1S mediated multidrug efflux is critical for survival of Xf in grapevines.  In Xf, T1S depends on a single TolC, possibly making this system more vulnerable to inhibition by small molecule treatments than those found in most bacterial pathogens, which typically encode redundant functional TolC variants.  Xf T1S mutants are much more sensitive to the surfactant Silwet L-77 than wild type Xf, and high throughput fluorescence and optical density assays of Xf cell viability were performed both with and without Silwet L-77.  GFP-marked Xf strain Temecula1 was used to screen two Prestwick combinatorial small molecule libraries (phytochemical and FDA approved drugs; 1600 chemicals in total) to assay Xf cell growth inhibition. Significant growth inhibition of Xf was observed with 209 chemicals, 150 of which exhibited growth inhibition enhanced by Silwet L-77, indicating these chemicals possibly target T1S efflux. Over a dozen natural antibiotics and phytochemicals were identified as strongly inhibitory (>100%) at 50 µM, including the phytoalexin gossypol and alkaloids berberine, remerine and olivicine.  Interestingly, the alkaloid harmalol significantly promoted Xf growth (2.28 X).  A dose-dependent effect of selected chemicals and sap and crude extracts from PD resistant grapevines on Xf cell viability and T1S efflux is underway.

Abstract Number: C3-3, P20-717
Session Type: Concurrent